The essential process of somatic gene therapy is the ability to perform gene transfer. In this process, recombinant genes are introduced into selected somatic cells. In its simplest form, gene transfer can be performed by simply injecting minute amounts of DNA into the nucleus of a cell. This process is called microinjection (1). Once recombinant genes are introduced into a cell, they can be recognized by the cells normal mechanisms for transcription, and a gene product will be expressed. The utility of microinjection for gene therapy is obviously limited by the number of cells which can be injected. Thus, more efficient methods have been developed for introducing DNA into larger numbers of cells. These methods include: transfection, where DNA is precipitated with CaPO.sub.4 and taken into cells by pinocytosis (2); electroporation, where cells are exposed to large voltage pulses to introduce holes into the membrane (3); lipofection/liposome fusion, where DNA is packaged into lipophilic vesicles which fuse with a target cell (4); and particle bombardment using DNA bound to small projectiles (5). Another important method for introducing DNA into cells is to couple the DNA to chemically modified proteins. These modified proteins have the ability to bind DNA through a chemically attached synthetic polylysine peptide, and bind to specific receptors on target cells. After these complexes are taken up by a specific receptor mediated endocytosis, the genes encoded by the DNA can be expressed by the target cell. Experiments have been performed with transferrin/polylysine/DNA complexes (6-11) as well as with asialoglycoprotein/polylysine/DNA complexes (12-25). The covalently, chemically combined natural ligands are used: (1) to specifically targeted DNA to different tissues; (2) to provide more efficient uptake process. These methods are limited because they require in vitro modification of the ligands using chemical or enzymatic methods in order to create a compound capable of binding DNA.
An important advance in methods of gene transfer was the demonstration that adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells. The admixture of adenovirus to solutions containing DNA complexes, or the binding of DNA to adenoviral particles by polylysine covalently attached to the adenovirus using protein crosslinking agents substantially improves the uptake and expression of the recombinant gene (6). Further studies have also suggested that proteins purified from other pathogenic bacteria, viruses or parasites may have similar properties.
In vivo studies with asialo-orosomucoid/polylysine/DNA complexes have shown that it is possible to achieve short term expression (several days) of recombinant genes in organs such as the liver (12-15). For this expression to be useful, however, it will be necessary to administer the complex repetitively either to treat intermittent symptoms of disease, or to establish chronic, steady-state levels of the gene product. One problem with repetitive administration using the asialo-orosomucoid/polylysine/DNA complex method, however, is that this complex is highly antigenic and that anaphylaxis can result from repetitive administration. Significantly, antibodies are formed against both the covalently modified orosomucoid as well as polylysine. Presumably, the natural protein (orosomucoid) which is not normally antigenic, is rendered antigenic by the covalent addition of polylysine. (It is less likely that removal of sialic acid makes this protein antigenic since this is a normal intermediate in synthesis and degradation of the protein.)
The present invention describes an improved method for gene transfer which enables specific targeting of DNA, enhanced uptake in various cell types and endosomal destabilization without the need to produce and purify viral proteins and without the need for covalent, chemical or enzymatic modifications to couple the DNA to the ligand. The present invention thus provides a method for gene transfer which will be easier and safer than currently available methods.